An Apoplastic Defensin of Wheat Elicits the Production of Extracellular Polysaccharides in Snow Mold

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Article An Apoplastic Defensin of Wheat Elicits the Production of Extracellular Polysaccharides in Snow Mold

Ayako Isobe 1,2, Chikako Kuwabara 1, Michiya Koike 1, Keita Sutoh 1, Kentaro Sasaki 1,3 and Ryozo Imai 1,3,*

1 Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Toyohira-ku, Sapporo 062-8555, Japan; [email protected] (A.I.); [email protected] (C.K.); [email protected] (M.K.); [email protected] (K.S.); [email protected] (K.S.) 2 Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan 3 Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 3-1-3 Kannondai, Tsukuba 305-8604, Japan * Correspondence: [email protected]; Tel.: +81-29-838-8378

Abstract: TAD1 (Triticum aestivum defensin 1) is a plant defensin specifically induced by low temper- ature in winter wheat. In this study, we demonstrated that TAD1 accumulated in the apoplast during cold acclimation and displayed antifungal activity against the pink snow mold fungi Microdochium nivale. When M. nivale was treated with TAD1, Congo red-stainable extracellular polysaccharides (EPS) were produced. The EPS were degradable by cellulase treatment, suggesting the involvement of β-1,4 glucans. Interestingly, when the fungus was treated with FITC-labeled TAD1, fluorescent signals were observed within the EPS layer. Taken together, these results support the hypothesis that the EPS plays a role as a physical barrier against antimicrobial proteins secreted by plants. We antici- pate that the findings from our study will have broad impact and will increase our understanding of plant–snow mold interactions under snow.

Citation: Isobe, A.; Kuwabara, C.; Keywords: apoplast; cold acclimation; defensin; extracellular polysaccharides; wheat Koike, M.; Sutoh, K.; Sasaki, K.; Imai, R. An Apoplastic Defensin of Wheat Elicits the Production of Extracellular Polysaccharides in Snow Mold. Plants 1. Introduction 2021, 10, 1607. https://doi.org/ Overwintering cereals and perennial grasses in temperate and boreal climate zones 10.3390/plants10081607 must endure prolonged exposure to subzero temperatures. The freezing tolerance of plants substantially increases after a period of exposure to low but non-freezing temperatures, Academic Editor: Aziz Aziz which is a well characterized process known as cold acclimation [1]. In northern areas with deep and persistent snow, winter cereals such as wheat and rye often suffer from Received: 17 July 2021 snow mold diseases caused by fungi such as Microdochium nivale, Typhula ishikariensis, and Accepted: 3 August 2021 Published: 5 August 2021 Sclerotinia borealis, while they are protected from freezing under the snow cover. It has been reported that resistance against snow mold fungus increases during cold acclimation in

Publisher’s Note: MDPI stays neutral overwintering plants such as winter wheat [2], barley [3], and Arabidopsis [4]. However, with regard to jurisdictional claims in the mechanism of cold-induced resistance to pathogens is poorly understood. published maps and institutional affil- In order to better understand this process and elucidate the functional mechanisms of iations. this phenomenon, we aimed to clone and functionally characterize novel defense-related genes that are induced during cold acclimation. We identified a plant defensin gene, TAD1 (Triticum aestivum defensin 1), that is specifically induced by cold treatment in winter wheat [5]. Plant defensins are known as a class of the pathogenesis-related (PR) proteins [6]. Under in vitro conditions, the recombinant TAD1 protein showed inhibitory Copyright: © 2021 by the authors. Pseudomonas cichorii T. ishikarien- Licensee MDPI, Basel, Switzerland. activity against the plant pathogen and the snow mold This article is an open access article sis [5,7]. Furthermore, overexpression of TAD1 confers resistance against T. ishikariensis in distributed under the terms and wheat [7]. TAD1 is induced by cold and abscisic acid treatment but not by defense-related conditions of the Creative Commons phytohormones such as salicylic acid and methyl jasmonate, suggesting that the gene is Attribution (CC BY) license (https:// under regulation of abiotic signals rather than biotic ones [5]. creativecommons.org/licenses/by/ Here, we report that the TAD1 protein accumulates in apoplasts of wheat leaf tissue 4.0/). during cold acclimation and suggest its involvement in cold-induced resistance against

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phytohormones such as salicylic acid and methyl jasmonate, suggesting that the gene is

Plants 2021, 10, 1607 under regulation of abiotic signals rather than biotic ones [5]. 2 of 8 Here, we report that the TAD1 protein accumulates in apoplasts of wheat leaf tissue during cold acclimation and suggest its involvement in cold-induced resistance against snow molds. We found that M. nivale secretes extracellular polysaccharides in response to TAD1snow treatment, molds. We providing found that functionalM. nivale secretesevidence extracellular for the existence polysaccharides of a defense in mechanism response to ofTAD1 the pathogen treatment, against providing host defense functional systems. evidence for the existence of a defense mechanism of the pathogen against host defense systems. 2. Results 2. Results 2.1.2.1. TAD1 TAD1 Localized Localized in in Extracellular Extracellular Space Space during during Cold Cold Acclimation Acclimation SinceSince TAD1 TAD1 has has putative putative N N-terminal-terminal signal signal peptides peptides [5], [5], it it was was expected expected that that TAD1 TAD1 entersenters into into the the secretary secretary pathway. pathway. We We analyzed analyzed the thesubcellular subcellular localization localization and andaccumu- accu- lationmulation of TAD1 of TAD1 during during cold coldacclimation. acclimation. An immunoblot An immunoblot analysis analysis with polyclo with polyclonalnal anti- bodiesantibodies against against TAD1 TAD1 did not did detect not detect any cross any cross-reacting-reacting bands bands in the in total the totalsoluble soluble fraction frac- (Figuretion (Figure S1). In S1 contrast,). In contrast, a cross a cross-reacting-reacting band band with with a molecular a molecular mass mass of approximately of approximately 6 kDa6 kDa was was detected detected in the in theapoplast apoplast fractions fractions (Figure (Figure 1), which1), which is close is close in size in sizeto that to of that ma- of turemature TAD1 TAD1 protein protein (5.54 (5.54 kDa). kDa). Accumulation Accumulation of the of the TAD1 TAD1 protein protein in in the the apoplast apoplast fraction fraction waswas detectable detectable at at the the first ﬁrst day day of of cold cold acclimation acclimation and and further further accumulated accumulated during during 14 14 d d of of coldcold acclimation acclimation (Figure (Figure 1B).1B). This This pattern pattern of of accumulat accumulationion was was in in good good accordance accordance with with thatthat of of TAD1TAD1 expressionexpression during during cold cold acclimation acclimation [5]. [5].

FigureFigure 1. 1. WesternWestern blot analysisanalysis ofof cold-acclimated cold-acclimated wheat wheat apoplastic apoplastic protein protein extracts. extracts. (A,B ()A Apoplastic,B) Apo- plasticproteins proteins from a from time a course time course of cold of treatment cold treatment were separated were separated by SDS-PAGE by SDS (A-PAGE), and a(A duplicated), and a du- gel plicatedwas transferred gel was transferred to a membrane to a membrane for Western for blot Western analysis blot (B ).analysis CA, cold (B acclimation.). CA, cold acclimation.

2.2.2.2. Subcellular Subcellular Localization Localization of of TAD1 TAD1-GFP-GFP Protein Protein TheThe subcellular subcellular localization localization of of TAD1 TAD1 was was also also analyzed analyzed using using GFP GFP-fusion-fusion proteins. proteins. TwoTwo ex expressionpression vectors vectors containing containing either either an an N- N-terminalterminal signal signal sequence sequence (SigTAD1) (SigTAD1) or ora fulla full length length sequence sequence of of TAD1 TAD1 fused fused with with synthetic synthetic green green fluorescent fluorescent protein protein (GFP) (GFP) (35S::SigTAD1(35S::SigTAD1-GFP-GFP or or 35S::TAD1 35S::TAD1-GFP)-GFP) were were introduced introduced to toand and transiently transiently expressed expressed in onionin onion epid epidermalermal cells. cells.Localization Localization of the proteins of the proteins was observed was observed with fluorescent with fluorescent microscopy.microscopy. When the When onion the cells onion were cells bombarded were bombarded with the with 35S::GFP the 35S::GFP vector as vector a control, as a control,green fluorescencegreen fluorescence was observed was observed in the cytoplasm in the cytoplasm and nucleus and nucleus (Figure (Figure 2A). Onion2A). Onion cells cellsthat werethat b wereombarded bombarded with either with either35S::TAD1 35S::TAD1-GFP-GFP or 35S::SigTAD1 or 35S::SigTAD1-GFP-GFP displayed displayed GFP fluo- GFP rescencefluorescence,, which which localized localized in the inouter the outerlayer of layer the ofcell the (Figure cell (Figure 2B,C).2 B,C).Since Sincethere therewas little was differencelittle difference between between TAD1 TAD1-GFP-GFP and andSigTAD1 SigTAD1-GFP,-GFP, it was it was concluded concluded that that the the subcellular subcellular locallocalizationization of of TAD1 TAD1 is is determined determined by by its its N N-terminal-terminal signal signal sequence. sequence. To address the TAD1 localization more in detail, we co-expressed an ER marker, DsRed-HDEL, with TAD1-GFP (Figure2D,E). DsRed-HDEL showed red fluorescence in a typical ER network, including nuclear and plasma membranes (Figure2E). A merged image clearly showed that TAD1-GFP fluorescence was detected in the outer layer of the cells, suggesting that the TAD1-GFP protein localizes to the cell wall and apoplast (Figure2F).

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FigureFigure 2. 2. SubcellularSubcellular localization localization of GFPGFP fusionfusion proteinsproteins in in onion onion epidermal epidermal cells. cells. Fluorescent Fluorescent images im- agesof the of cellsthe cells transformed transformed with with GFP GFP vector vector (A), (A SigTAD1-GFP), SigTAD1-GFP (B), (B and), and TAD1-GFP TAD1-GFP (C ).(C Scale). Scale bar bar for forA =A20 = 20µm µm; scale; scale bar bar for for B and B and C = C 50 =µ 50m. µm. (D, E(D) Apoplastic,E) Apoplastic localization localization of the of TAD1 the TAD1 protein protein in onion in onionepidermal epidermal cells. cells. Fluorescent Fluorescent images images of the of cells the co-bombarded cells co-bombarded with TAD1-GFP with TAD1 (D-GFP) and (D an) and ER markeran ER marker DsRed-HDEL (E). (F) A merged image of D and E. Scale bar = 10 µm. DsRed-HDEL (E). (F) A merged image of D and E. Scale bar = 10 µm.

2.3.To TAD1 address Induced the Production TAD1 localization of Extracellular more Polysaccharides in detail, we in co M.-expressed nivale an ER marker, DsRedAntifungal-HDEL, with activities TAD1 of-GFP the recombinant(Figure 2D,E). mature DsRed TAD1-HDEL (rmTAD1) showed against red fluorescence the pink snow in amold, typicalM. ER nivale network,, were including determined nuclear by hyphal and plasma growth membranes inhibition. As(Figure shown 2E). in AFigure merge3Ad , imagegrowth clearly of M. showed nivale was that inhibited TAD1-GFP by the fluorescence addition of was rmTAD1, detected indicating in the outer that TAD1layer of has the an cells,antifungal suggesting activity that against the TAD1M. nivale-GFP. protein When rmTAD1localizes wasto the added cell wall to the and culture apoplast of M. (Figure nivale , 2F).morphological changes of M. nivale were observed, including hyphal aggregation and hyphal tip swelling. In contrast, however, normal hyphal growth was observed without 2.3.the TAD1 addition Induce of thed Production protein (Figure of Extracellular3B). These Polysaccharides changes caused in M. by n rmTAD1ivale suggested that TAD1Antifungal has a function activities to make of the fungus recombinant cell division mature anomalously. TAD1 (rmTAD1) More surprisingly, against the pink treat- snowment mold, with rmTAD1M. nivale induced, were determined the accumulation by hyphal of extracellulargrowth inhibition. substances As shown around in hyphae.Figure 3A,Subsequent growth of staining M. nivale with was Congo inhibited red by suggested the addition that theof rmTAD1, substances indicating contain polysaccha-that TAD1 hasrides an (Figureantifungal3C ). activity Since it against has been M. previously nivale. When reported rmTAD1 that wasM. nivale addeduniquely to the culture produces of M.extracellular nivale, morphological cellulose [8 changes], it is plausible of M. nivale that thewere produced observed, extracellular including hyphal polysaccharides aggregation(EPS) and are hyphal cellulose. tip swelling. These results In contrast, suggested however, that M. normal nivale produceshyphal growth EPS in was response observed to a withoutplant defense the addition protein of TAD1. the protein (Figure 3B). These changes caused by rmTAD1 sug- gestedIn that some TAD1 bacteria, has a EPSfunction are involved to make infungus the protection cell division against anomalously. chemical More and biological surprisingly,attacks treatment [9,10]. Consequently, with rmTAD1 we induced therefore the tested accumulation the possibility of extracellularthat EPS produced substances by M. aroundnivale has hyphae. a protective Subsequent role againststaining the with plant Congo defense red suggested system. To that address the substances the interaction con- tainbetween polysaccharides TAD1 and EPS, (Figure welabeled 3C). Since rmTAD1 it has with been fluorescein previously isothiocyanate reported that (FITC). M. nivale Func- uniquelytionality prod of theuces rmTAD1-FITC extracellular protein cellulose was [8], confirmed it is plausible by observing that the produced growth inhibition extracellular and polysaccharidesEPS production (EPS) in M. are nivale cellulose.(Figure These4A). Fluorescentresults suggested images that of M. rmTAD1-FITC nivale produces demon- EPS instrated response that to rmTAD1-FITC a plant defense accumulated protein TAD1. toa high level within the EPS layer (Figure4B). Subsequent treatment with 2% cellulase decreased the levels of EPS around the hyphae (Figure4C) and concomitantly diminished fluorescence signals (Figure4D). These data suggested that the EPS exhibits some affinity to TAD1. This observation supported a putative function of EPS produced by M. nivale in the protection from the host plant defense system.

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FigureFigure 3. 3. AntifungalAntifungal activity activity of of TAD1 TAD1 to to snow snow mold mold fungus fungus M.M. nivale nivale. .((AA)) Relative Relative fungus fungus growth growth waswas estimated estimated by by cultivation cultivation of of the the M.M. nivale nivale hyphaehyphae on on a a 96 96-well-well microplate microplate and and increasing increasing the the concentrationconcentration of of rmTAD1 rmTAD1 (0, (0, 2, 2,10 10,, and and 20 20µg/mL)µg/mL) at 10 at °C 10 for◦C 9 fordays. 9 days. The hyphal The hyphal growth growth was mon- was itoredmonitored by measuring by measuring absorbance absorbance at 595 at 595nm nmand and was was presented presented as asa percentage a percentage of ofthe the growth growth in in the the absence of rmTAD1. Two biological replicate experiments were performed (experiment one and absence of rmTAD1. Two biological replicate experiments were performed (experiment one and two). two). (B,C) Microscopic observation of M. nivale hyphae after incubating spores at 10 °C for 9 days (B,C) Microscopic observation of M. nivale hyphae after incubating spores at 10 ◦C for 9 days with or Plants 2021, 10, x FOR PEER REVIEWwith or without rmTAD1 (10 µg/mL). Images of hyphae were captured by using differential inter-5 of 9 µ ferencewithout contrast rmTAD1 (DIC) (10 microscopyg/mL). Images (B). Mycelia of hyphae were were stained captured with Congo by using red differential (C). Scale bar interference = 20 µm. contrast (DIC) microscopy (B). Mycelia were stained with Congo red (C). Scale bar = 20 µm. In some bacteria, EPS are involved in the protection against chemical and biological attacks [9,10]. Consequently, we therefore tested the possibility that EPS produced by M. nivale has a protective role against the plant defense system. To address the interaction between TAD1 and EPS, we labeled rmTAD1 with fluorescein isothiocyanate (FITC). Functionality of the rmTAD1-FITC protein was confirmed by observing growth inhibition and EPS production in M. nivale (Figure 4A). Fluorescent images of rmTAD1-FITC demonstrated that rmTAD1-FITC accumulated to a high level within the EPS layer (Figure 4B). Subsequent treatment with 2% cellulase decreased the levels of EPS around the hyphae (Figure 4C) and concomitantly diminished fluorescence signals (Figure 4D). These data suggested that the EPS exhibits some affinity to TAD1. This observation supported a putative function of EPS produced by M. nivale in the protection from the host plant defense system.

FigureFigure 4. 4. TAD1TAD1 accumulated accumulated to to a ahigh high level level within within the the EPS EPS layer. layer. Microscopic Microscopic observation observation of of M.M. nivalenivale mymyceliacelia after after incubating incubating spores spores at at 15 15 °C◦C for for 7 7 days days with with FITC FITC-labelled-labelled rmTAD1 rmTAD1 (10 (10 µg/mL)µg/mL) throughthrough bright bright field field and and FITC FITC filter filter sets. sets. The The first first row row (A (A,B,B) represents) represents the the images images without without cellulase. cellulase. The second row (C,D) represents the images with cellulase. The second row (C,D) represents the images with cellulase.

3. Discussion Overwintering plants are known to acquire disease resistance during cold acclimation [2,4]. This mechanism is distinct from other induced defense mechanisms of plants in that it proceeds prior to infection and is considered as a preventative adaptation. Here,

we demonstrated that TAD1 accumulates in the apoplast during cold acclimation (Figures 1 and 2) and exhibits an antifungal activity against M. nivale (Figure 3A,B). It is known that apoplast is the extracellular compartment, which is a major battlefield of plants and pathogens [11]. Therefore, it is reasonable to consider that host plants secrete antimicrobial proteins to the apoplast as a defense response [12]. Our data suggested that TAD1 is involved in such a defense mechanism, although its accumulation is cold-induced and preventive (Figure 5).

Low temp.

TAD1

Wheat Snow mold

Step 1: Winter wheat produces TAD1 in response to cold Step 2: Secreted TAD1 inhibits fungal growth and protects the plant Step 2: M. nivale recognizes TAD1 and produces EPS Step 3: The EPS protect the fungus from the plant defense protein

Figure 5. A proposed model for interaction of defense mechanisms between wheat and M. nivale.

We observed EPS production in M. nivale in response to TAD1 and detected colocal- ization of TAD1 with the EPS (Figures 3C and 4). Collectively, these data suggest a function of the EPS as a protective barrier from antifungal proteins (Figure 5). A similar phenomenon was reported for Magnaporthe grisea, where polysaccharide α-1,3-glucan is induced by plant wax and protects the hyphal cell wall from digestive enzymes produced by plants [13]. In addition, it was also demonstrated that the activity of human antimicrobial peptides was suppressed in the presence of polysaccharides produced by lung pathogens [14]. We hypothesized that M. nivale produces EPS to protect its hyphae from plant antimicrobial proteins through direct interaction. It is not known, however, how M. nivale

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Figure 4. TAD1 accumulated to a high level within the EPS layer. Microscopic observation of M. nivale mycelia after incubating spores at 15 °C for 7 days with FITC-labelled rmTAD1 (10 µg/mL) Plants 2021, 10, 1607 through bright field and FITC filter sets. The first row (A,B) represents the images without cellulase.5 of 8 The second row (C,D) represents the images with cellulase.

3. Discussion 3. Discussion Overwintering plants are known to acquire disease resistance during cold acclima- Overwintering plants are known to acquire disease resistance during cold accli- tion [2,4]. This mechanism is distinct from other induced defense mechanisms of plants in mation [2,4]. This mechanism is distinct from other induced defense mechanisms of thatplants it proceeds in that it prior proceeds to infection prior to and infection is considered and is considered as a preventative as a preventative adaptation. adaptation. Here, weHere, demonstrated we demonstrated that TAD1 that accumulates TAD1 accumulates in the apoplast in the during apoplast cold during acclimation cold acclimation (Figures 1 (andFigures 2) and1 and exhibits2 ) and an exhibits antifungal an antifungalactivity against activity M. againstnivale (FigureM. nivale 3A,(FigureB). It is3 knownA,B). It thatis knownapoplast that is the apoplast extracellular is the extracellular compartment compartment,, which is a major which ba isttlefield a major of battleﬁeldplants and of pathogensplants and [11]. pathogens Therefore, [11 ].it Therefore,is reasonable it isto reasonable consider that to consider host plants that secrete host plants antimicro- secrete bialantimicrobial proteins to proteinsthe apoplast to the as apoplast a defense as response a defense [12]. response Our data [12]. suggested Our data suggestedthat TAD1 thatis involvedTAD1 is in involved such a indefense such a mechanism, defense mechanism, although although its accumulation its accumulation is cold- isinduced cold-induced and preventiveand preventive (Figure (Figure 5). 5).

Low temp.

TAD1

Wheat Snow mold

Figure 5. A proposed model for interaction of defense mechanisms between wheat and M. nivale. Figure 5. A proposed model for interaction of defense mechanisms between wheat and M. nivale. We observed EPS production in M. nivale in response to TAD1 and detected colocaliza- We observed EPS production in M. nivale in response to TAD1 and detected colocal- tion of TAD1 with the EPS (Figures3C and4). Collectively, these data suggest a function of ization of TAD1 with the EPS (Figures 3C and 4). Collectively, these data suggest a func- the EPS as a protective barrier from antifungal proteins (Figure5). A similar phenomenon tion of the EPS as a protective barrier from antifungal proteins (Figure 5). A similar phe- was reported for Magnaporthe grisea, where polysaccharide α-1,3-glucan is induced by plant nomenonwax and was protects reported the hyphalfor Magnaporthe cell wall fromgrisea digestive, where polysaccharide enzymes produced α-1,3-glucan by plants is in- [13]. ducedIn addition, by plant it wax was and also protects demonstrated the hyphal that thecell activitywall from of humandigestive antimicrobial enzymes produced peptides bywas plants suppressed [13]. In addition, in the presence it was also of polysaccharides demonstrated that produced the activity by lung of human pathogens antimicro- [14]. We bialhypothesized peptides was that suppressedM. nivale producesin the presence EPS to of protect polysaccharides its hyphae produced from plant by antimicrobial lung path- ogensproteins [14]. throughWe hypothesized direct interaction. that M. nivale It is produces not known, EPS however, to protect how its hyphaeM. nivale fromperceives plant antimicrobialthe presence proteins of antimicrobial through direct proteins. interaction. In plants, It pathogen-derivedis not known, however, molecules, how M. known nivale as microbe-associated molecular patterns (MAMPs), are perceived as signals that triggers induction of immune responses [15]. In addition, endogenous molecules released from damaged cells after pathogen attack are recognized as danger signals [15,16]. It is reasonable to consider that similar mechanisms may be possible for the pathogens to recognize host defense mechanisms. Physiological changes caused by TAD1 may trigger a signal that induces the production of EPS. On the other hand, it is also possible that TAD1 can be recognized as a molecular pattern. Although plant–pathogen interactions have been extensively studied, very few studies have focused on the pathogen’s responses to plant defense systems. EPS production in response to TAD1 treatment may be utilized as a model system to decipher this novel plant–microbe interaction.

4. Conclusions Wheat accumulates an apoplastic defensin, TAD1, during cold acclimation as a preventive response against snow molds. The pink snow mold, M. nivale, counteract the plant defense by producing EPS that capture TAD1. Plants 2021, 10, 1607 6 of 8

5. Materials and Methods 5.1. Plant Growth and Cold Acclimation Conditions Seeds of winter wheat (Triticum aestivum L. cv. Chihokukomugi) were sown in commer- cial potting mix and grown in a growth chamber that was maintained under 20 ◦C/15 ◦C (16 h light/8 h dark) cycles for 3 weeks. Cold acclimation was conducted by transferring plants to 2 ◦C (10 h light/14 h dark) in an environmentally controlled growth room. Plants were harvested prior to and during 14 d of cold treatment. The light intensity was set to 256 µmol m−2 s−1.

5.2. Extraction of Apoplastic Proteins Apoplastic proteins were extracted as previously described by Hon et al. [17], with slight modifications. Leaf blades (50 g) of non-acclimated and cold-acclimated wheat plants were harvested and cut into 3 cm segments. These segments were gathered and tied, and the cut surface was washed with deionized water. Subsequently, the leaf segments were soaked in an extraction buffer (20 mM ascorbic acid, 20 mM CaCl2, pH 3.0) and vacuum infiltrated for 30 min. The leaf segments were then surface-dried with a paper towel and then placed in a plastic syringe barrel (10 mL). Afterwards, the syringe barrel was inserted into a 15 mL conical tube. Apoplastic fluids were recovered by centrifugation at 2000× g for 10 min using a swing out rotor. The fluid collected at the bottom of the tube was subsequently concentrated with a Microcon YM-3 (Millipore, Burlington, MA, USA). Protein concentration was estimated by using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and BSA was used as a standard.

5.3. Western Blot Analysis Extracted apoplastic protein (4 µg) or soluble protein (25 µg) fractions were separated by SDS-PAGE and transferred onto Hybond-C extra membrane (GE Healthcare, Chicago, IL, USA). Hybridization was performed with primary polyclonal rabbit serum, which was raised against recombinant mature TAD1 (1:1000 v/v) and anti-rabbit IgG peroxidase-linked secondary antibody (1:10,000 v/v) (GE Healthcare, Chicago, IL, USA). Chemiluminescent detection of the signal was carried out with the ECL kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions.

5.4. Localization of GFP-Fused Protein The entire coding sequence and the putative signal sequence (SigTAD1) of TAD1 was fused with a synthetic green fluorescent protein, sGFP (S65T) [18], and utilized as a reporter gene for TAD1 localization. As a reference, we used a marker gene for ER localization (DsRed-HDEL) [19,20]. Onion (Allium cepa) bulb scales were cut into small squares and placed onto a Murashige and Skoog (MS) agar plate (pH 8.4). TAD1-GFP, SigTAD1-GFP, and DsRed-HDEL plasmids were delivered into onion epidermal cells using particle bombardment. Gold particles (1.0 µm; Bio-Rad, Hercules, CA, USA) were coated with plasmid DNA according to the manufacturer’s instructions. Particles were bombarded into the onion epidermal cells using a Biolistic PDS-1000/He system (Bio-Rad, Hercules, CA, USA) with 1100 psi rupture discs that were operated under a vacuum condition. After bombardment, cells were allowed to recover for 18–22 h on the MS agar medium at 25 ◦C in the dark. Expression of the fusion constructs was monitored by using a Leica fluorescent microscopic system (FW4000). The filter sets used were the GFP filter (excitation; 470 nm, emission; 525 nm) for GFP fusion protein and the RFP filter (excitation; 546 nm, emission; 605 nm) for DsRed-HDEL protein, respectively.

5.5. Recombinant Protein Purification Purification of recombinant mature TAD1 protein fused with GST (GST-mTAD1) was carried out as previously described [5]. GST-mTAD1 was induced by the treatment with IPTG (0.5 mM) for 4 h at 30 ◦C. Soluble proteins were recovered with the BugBuster® Protein Extraction Reagent (Merck, Darmstadt, Deutschland). Affinity purification of the Plants 2021, 10, 1607 7 of 8

recombinant protein was performed by using a glutathione-sepharose 4B column (GE Healthcare, Chicago, IL, USA) and GST-mTAD1 was digested with PreScission® Protease (GE Healthcare, Chicago, IL, USA) for 8 h at 4 ◦C according to the supplier’s instructions. The puriﬁed rmTAD1 was concentrated by a Microcon YM-3 column (Millipore, Burlington, MA, USA), and the concentration was determined with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as a standard.

5.6. Antifungal Activity of TAD1 Antifungal activity was analyzed as described previously, with slight modiﬁcations [21]. M. nivale (MCW222-7) [22] was cultured on potato dextrose agar (PDA; BD, Franklin Lakes, NJ, USA) medium at 4 ◦C for one month. Potato dextrose medium (20 mL) was inoculated with one agar plug (5 mm in a diameter) of M. nivale grown on PDA medium and cultured at 10 ◦C for 5 days with shaking at 120 rpm. The culture was crushed twice for 15 s with a polytron homogenizer (Kinematica AG, Littau, Switzerland). To test the hyphal growth inhibitory effect of TAD1, we cultured fragmented hyphae with 0, 2, 10, and 20 µg/mL rmTAD1 for approximately 9 days at 10 ◦C in a 96-well microtiter plate. Growth was measured by determining optical density at 595 nm in a spectrophotometer. To test the inhibitory effect of TAD1 against spore germination, we cultured M. nivale on PDA medium at 10 ◦C for one month under a black light lamp. After one month, spores were collected with water and 20 µL of spore suspension (1 × 10−6 conidia mL−1) was mixed with 40 µL of PBS or rmTAD1 solution in PBS and 140 µL potato dextrose medium in a 96-well plate. After a 9 day incubation at 10 ◦C, the mixtures were stained with 10 µL of 1% Congo red solution, subsequently washed three times with distilled water, and observed through a differential interference contrast (DIC) microscope.

5.7. FITC Labeling Labeling of rmTAD1 with FITC (Wako Pure Chemical industries, Osaka, Japan) was performed according to the manufacturer’s instructions.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/plants10081607/s1, Figure S1: Western blot analysis of cold-acclimated wheat total protein extracts. Author Contributions: Conceptualization, R.I.; design of the experiments, C.K., M.K., and R.I.; conduction of the experiments, A.I., C.K., and M.K.; microscopic analysis, K.S. (Keita Sutoh); data analysis, K.S. (Kentaro Sasaki) and R.I.; writing—original draft preparation, review and editing, K.S. (Kentaro Sasaki) and R.I. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries (Biodesign #1207). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article. Acknowledgments: We would like to thank our group members at the Hokkaido Agricultural Research Center and the Institute of Agrobiological Sciences of NARO for their supports. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References 1. Guy, C.L. Cold acclimation and freezing stress tolerance: Role of protein metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990, 41, 187–223. [CrossRef] 2. Nakajima, T.; Abe, J. Environmental factors affecting expression of resistance to pink snow mold caused by Microdochium nivale in winter wheat. Can. J. Bot. 1996, 74, 1783–1788. [CrossRef] 3. Tronsmo, A.M.; Gregersen, P.; Hjeljord, L. Cold-Induced Disease Resistance. In Mechanisms of Plant Defense Responses; Springer: Berlin/Heidelberg, Germany, 1993; p. 369. Plants 2021, 10, 1607 8 of 8

4. Kuwabara, C.; Sasaki, K.; Umeki, N.; Hoshino, T.; Saburi, W.; Matsui, H.; Imai, R. A model system for studying plant–microbe interactions under snow. Plant Physiol. 2021, 185, 1489–1494. [CrossRef][PubMed] 5. Koike, M.; Okamoto, T.; Tsuda, S.; Imai, R. A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation. Biochem. Biophys. Res. Commun. 2002, 298, 46–53. [CrossRef] 6. Ali, S.; Ganai, B.A.; Kamili, A.N.; Bhat, A.A.; Mir, Z.A.; Bhat, J.A.; Tyagi, A.; Islam, S.T.; Mushtaq, M.; Yadav, P.; et al. Pathogenesis- related proteins and peptides as promising tools for engineering plants with multiple stress tolerance. Microbiol. Res. 2018, 212–213, 29–37. [CrossRef][PubMed] 7. Sasaki, K.; Kuwabara, C.; Umeki, N.; Fujioka, M.; Saburi, W.; Matsui, H.; Abe, F.; Imai, R. The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat. J. Biotechnol. 2016, 228, 3–7. [CrossRef] 8. Schweiger-Hufnagel, U.; Ono, T.; Izumi, K.; Hufnagel, P.; Morita, N.; Kaga, H.; Morita, M.; Hoshino, T.; Yumoto, I.; Matsumoto, N.; et al. Identification of the extracellular polysaccharide produced by the snow mold fungus. Microdochium nivale. Biotechnol. Lett. 2000, 22, 183–187. [CrossRef] 9. François, F.; Lombard, C.; Guigner, J.M.; Soreau, P.; Brian-Jaisson, F.; Martino, G.; Vandervennet, M.; Garcia, D.; Molinier, A.L.; Pignol, D.; et al. Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury. Appl. Environ. Microbiol. 2012, 78, 1097–1106. [CrossRef] 10. Aslam, S.N.; Newman, M.A.; Erbs, G.; Morrissey, K.L.; Chinchilla, D.; Boller, T.; Jensen, T.T.; De Castro, C.; Ierano, T.; Molinaro, A.; et al. Bacterial Polysaccharides Suppress Induced Innate Immunity by Calcium Chelation. Curr. Biol. 2008, 18, 1078–1083. [CrossRef] 11. Doehlemann, G.; Hemetsberger, C. Apoplastic immunity and its suppression by filamentous plant pathogens. New Phytol. 2013, 198, 1001–1016. [CrossRef] 12. Naz, R.; Bano, A.; Wilson, N.L.; Guest, D.; Roberts, T.H. Pathogenesis-related protein expression in the apoplast of wheat leaves protected against leaf rust following application of plant extracts. Phytopathology 2014, 104, 933–944. [CrossRef] 13. Fujikawa, T.; Kuga, Y.; Yano, S.; Yoshimi, A.; Tachiki, T.; Abe, K.; Nishimura, M. Dynamics of cell wall components of Magnaporthe grisea during infectious structure development. Mol. Microbiol. 2009, 73, 553–570. [CrossRef] 14. Benincasa, M.; Mattiuzzo, M.; Herasimenka, Y.; Cescutti, P.; Rizzo, R.; Gennaro, R. Activity of antimicrobial peptides in the presence of polysaccharides produced by pulmonary pathogens. J. Pept. Sci. 2009, 15, 595–600. [CrossRef] 15. Choi, H.W.; Klessig, D.F. DAMPs, MAMPs, and NAMPs in plant innate immunity. BMC Plant Biol. 2016, 16, 232. [CrossRef] [PubMed] 16. Tanaka, K.; Choi, J.; Cao, Y.; Stacey, G. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants. Front. Plant Sci. 2014, 5, 446. [CrossRef][PubMed] 17. Hon, W.-C.; Griffith, M.; Chong, P.; Yang, D.S.C. Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol. 1994, 104, 971–980. [CrossRef][PubMed] 18. Niwa, Y.; Hirano, T.; Yoshimoto, K.; Shimizu, M.; Kobayashi, H. Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. Plant J. 1999, 18, 455–463. [CrossRef][PubMed] 19. Okamoto, T.; Shimada, T.; Hara-Nishimura, I.; Nishimura, M.; Minamikawa, T. C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase. Plant Physiol. 2003, 132, 1892–1900. [CrossRef] 20. Nakaminami, K.; Karlson, D.T.; Imai, R. Functional conservation of cold shock domains in bacteria and higher plants. Proc. Natl. Acad. Sci. USA 2006, 103, 10122–10127. [CrossRef] 21. Spelbrink, R.G.; Dilmac, N.; Allen, A.; Smith, T.J.; Shah, D.M.; Hockerman, G.H. Differential Antifungal and Calcium Channel- Blocking Activity among Structurally Related Plant Defensins. Plant Physiol. 2004, 135, 2055–2067. [CrossRef] 22. Christova, P.K.; Christov, N.K.; Imai, R. A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus. Microdochium nivale. Planta 2006, 223, 1207–1218. [CrossRef][PubMed]